It is desirable in certain circumstances to measure very low concentrations of certain organic compounds. In medicine, for example, it is very useful to determine the concentration of a given kind of molecule, usually in solution, which either exists naturally in physiological fluids (e.g. blood or urine) or which has been introduced into the living system (e.g. drugs or contaminants). Because of the rapidly advancing state of understanding of the molecular basis of both the normal and diseased states of living systems, there is an increasing need for methods of detection which are quantitative, specific to the molecule of interest, highly sensitive and relatively simple to implement. Examples of molecules of interest in a medical and/or biological context include, but are not limited to, drugs, sex and adrenal hormones, biologically active peptides, circulating hormones and antigens associated with tumors or infectious agents. In the case of drugs, for example, the safe and efficacious use of a particular drug requires that its concentration in the circulatory system be held to within relatively narrow bounds, referred to as the therapeutic range.
One broad approach used to detect the presence of a particular compound, referred to as the analyte, is the immunoassay, in which detection of a given molecular species, referred to generally as the ligand, is accomplished through the use of a second molecular species, often called the antiligand, or the receptor, which specifically binds to the first compound of interest. The presence of the ligand of interest is detected by measuring, or inferring, either directly or indirectly, the extent of binding of ligand to antiligand. The ligand may be either monoepitopic or poly epitopic and is generally defined to be any organic molecule for which there exists another molecule (i.e. the antiligand) which specifically bonds to said ligand, owing to the recognition of some portion of said ligand. Examples of ligands include macromolecular antigens and haptens (e.g. drugs). The antiligand, or receptor, is usually an antibody, which either exists naturally or can be prepared artificially. The ligand and antiligand together form a homologous pair. Throughout the text the terms antigen and antibody, which represent typical examples, are used interchangeably with the terms ligand and antiligand, respectively, but such usage does not signify any loss of generality. In some cases, the antibody would be the ligand and the antigen the antiligand, if the presence of the antibody is to be detected.
Implementation of a successful immunoassay requires a detectable signal which is related to the extent of antigen-antibody binding which occurs upon the reaction of the analyte with various assay reagents. Usually that signal is provided for by a label which is conjugated to either the ligand or the antiligand, depending on the mode of operation of the immunoassay. Any label which provides a stable, conveniently detectable signal is an acceptable candidate. Physical or chemical effects which produce detectable signals, and for which suitable labels exist, include radioactivity, fluorescence, chemiluminescence, phosphorescence and enzymatic activity, to name a few.
Broadly speaking, immunoassays fall into two general categories--heterogeneous and homogeneous. In heterogeneous assays, the purpose of the label is simply to establish the location of the molecule to which it conjugates--i.e. to establish whether the labeled molecule is free in solution or is part of a bound complex. Heterogeneous assays generally function by explicitly separating bound antigen-antibody complexes from the remaining free antigen and/or antibody. A method which is frequently employed consists of attaching one of the members of the homologous pair to a solid surface by covalent binding, physical absorption, or some other means. When antigen-antibody binding occurs, the resulting bound complexes remain attached to this solid surface (composed of any suitable inert material such as plastic, paper, glass, metal, polymer gel, etc.), allowing for separation of free antigen and/or antibody in the surrounding solution by a wash step. A variation on this method consists of using small (typically 0.05 to 20 microns) suspendable particles to provide the solid surface onto which either antigen or antibody is immobilized. Separation is effected by centrifugation of the solution of sample, reagents and suspendable beads at an appropriate speed, resulting in selective sedimentation of the support particles together with the bound complexes.
In the homogeneous assay, the signal obtained from the labeled ligand or antiligand is modified, or modulated, in some systematic, recognizable way when ligand-antiligand binding occurs. Consequently, separation of the labeled bound complexes from the free labeled molecules is no longer required.
There exist a number of ways in which immunoassays can be carried out.
In the competitive mode, the analyte, assumed to be antigen, is allowed to compete with a known concentration of labeled antigen (provided in reagent form in the assay kit) for binding to a limited number of antibody molecules which are attached to a solid matrix. Following an appropriate incubation period, the reacting solution is washed away, ideally leaving just labeled antigen-antibody complexes attached to the binding surface, thereby permitting the signal from the labels to be quantitated.
In another method, called the sandwich mode, the analyte, again assumed to be antigen, reacts with an excess of surface-immobilized antibody molecules. After a suitable incubation period, an excess of label-conjugated antibody is added to the system to react with another bonding site on the antigen. After this reaction has gone to essential completion, a wash step removes unbound labeled antibody and other sources of contamination, permitting measurement of the signal produced by labels which are attached to antibody-antigen-antibody complexes. Any non-specific bonding of the labeled antibody to the surface will, however, contribute to the signal.
In yet another approach, called the indirect mode, the analyte, this time assumed to consist of specific antibody, is allowed to bind to surface-immobilized antigen which is in excess. The binding surface is then washed and allowed to react with label-conjugated antibody. After a suitable incubation period the surface is washed again, removing free labeled antibody and permitting measurement of the signal due to bound labeled antibody. The resulting signal strength varies inversely with the concentration of the starting (unknown) antibody, since labeled antibody can bind only to those immobilized antigen molecules which have not already complexed to the analyte.
One of the most sensitive immunoassays developed thusfar is the radioimmunoassay (RIA), in which the label is a radionuclide, such as I.sup.125, conjugated to either member of the homologous (binding) pair.
Fluorescence provides a potentially attractive alternative to radioactivity as a suitable label for immunoassays. For example, fluorescein (usually in the form of fluorescein isothiocyanate, or "FITC") and a variety of other fluorescent dye molecules can be attached to most ligands and receptors without significantly impairing their binding properties. Fluorescent molecules have the property that they absorb light over a certain range of wavelengths and (after a delay ranging form 10-.sup.9 to 10-.sup.4 seconds) emit light over a range of longer wavelengths. Hence, through the use of a suitable light source, detector and optics, including excitation and emission filters, the fluorescence intensity originating from labeled molecules can be determined.
Use of an enzyme as a label has produced a variety of useful enzyme immunoassays (EIA), the most popular of which is known as ELISA. In the typical heterogeneous format a sandwich-type reaction is employed, in which the ligand of interest, assumed here to be antigen, binds to surface-immobilized specific antibody and then to an enzyme-antibody conjugate. After suitable incubation, any remaining free enzyme conjugate is eliminated by a wash or centrifugation step. A suitable substrate for the enzyme is then brought into contact with the surface containing the bound complexes. The enzyme-substrate pair is chosen to provide a reaction product which yields a readily detectable signal, such as a color change or a fluorescence emission. The use of an enzyme as a label serves to effectively amplify the contribution of a single labeled bound complex to the measured signal, because many substrate molecules can be converted by a single enzyme molecule.
As may be seen from the above background summary many of the immunoassay techniques rely on optical methods to detect specific ligands in a sample matrix and it may be said that these techniques have fallen generally into three optical classes:
1) Methods based on the molecular absorbance of light, which comprise all standard spectroscopic methods such as absorbance, fluorescence, phosphorescence, fluorescence polarization, circular dichroism, Raman, and infrared spectroscopies;
2) Methods based on the generation of light by a chemical reaction, which is known as chemiluminescence, or bioluminescence when the reaction is catalyzed by an enzyme; and
3) Methods based on the change in the direction of propagation of a lightwave such as refractometry, optical rotary dispersion, and methods based on the scattering of light.
With all of the above the signal is collected and measured by a detection apparatus which monitors the phenomenon as it occurs within the sample's molecular population. The measurement is a function of the intensity or degree of change occurring within the sample. Although most of these methods produce isotropic signals, even the anisotropic methods such as fluorescence polarization, produce signals which are fundamentally measured as intensities.
Emerging now are optical detection techniques which may comprise a new and fourth class of optical methods based on spatial patterns produced by the interaction of radiation with the ligand or anti-ligand. In this class of optical detection techniques the optical signal is collected outside of the sample, as a pattern or an intensity measured at more than one geometrically defined spatial position. The following descriptions are representative of this new class of optical detection methods.
Nicoli in U.S. Pat. No. 4,647,544 issued Mar. 3, 1987 entitled "Immunoassay Using Optical Interference Detection" describes optical detection of a binding reaction between a ligand and an antiligand wherein a pattern is formed on a substrate by a spatial array of microscopic dimensions of antiligand material immobilized to a substrate. Upon exposure to the sample the ligand binds to the substrate to form a physical pattern. A source of optical radiation is directed to the pattern at a particular incidence angle to produce an optical interference pattern in accordance with the binding reaction and with a strong scattering intensity at one or more Bragg scattering angles. An optical detector is located relative to the pattern and aligned with the Bragg scattering angle to detect the strong scattering intensity and produce a signal representative of the binding reaction.
Another patent, "Diffraction Immunoassay and Reagents" EPO 0276968 published Aug. 3, 1988 describes a "biograting" based assay in which a biological diffraction grating consisting of lines of active binding reagent is formed on a silicon substrate surface. After contacting the assay surface with the sample and separating the sample from the assay, the surface is illuminated and the binding of analyte to surface in a uniform manner generates a diffraction pattern. An optical detector, or array of detectors, positioned at predetermined angles is used to measure the diffracted light.
PCT Application No. PCT/GB85/00427 describes the use of fluorescently tagged molecules binding to a substrate pattern so that the fluorescence emission is organized into a narrow cone of angles instead of being uniform in all directions.
In each of these examples the detected signal is produced by light interacting with analytes that have been entrapped on a surface in a geometric manner, and the detectable signal is characterized by both amplitude and pattern formation.
These spatial based optical detection immunoassay methods are controlled by the spatial geometry, formed by either the ligand or antiligand, which must be of extremely fine detail and small dimensions; sophisticated immobilization technology will be required which may be difficult to reproducibly implement for many useful ligands or antiligands.